A TCF4-dependent gene regulatory network confers resistance to immunotherapy in melanoma.

To better understand intrinsic resistance to immune checkpoint blockade (ICB), we established a comprehensive view of the cellular architecture of the treatment-naive melanoma ecosystem and studied its evolution under ICB. Using single-cell, spatial multi-omics, we showed that the tumor microenvironment promotes the emergence of a complex melanoma transcriptomic landscape. Melanoma cells harboring a mesenchymal-like (MES) state, a population known to confer resistance to targeted therapy, were significantly enriched in early on-treatment biopsies from non-responders to ICB. TCF4 serves as the hub of this landscape by being a master regulator of the MES signature and a suppressor of the melanocytic and antigen presentation transcriptional programs. Targeting TCF4 genetically or pharmacologically, using a bromodomain inhibitor, increased immunogenicity and sensitivity of MES cells to ICB and targeted therapy. We thereby uncovered a TCF4-dependent regulatory network that orchestrates multiple transcriptional programs and contributes to resistance to both targeted therapy and ICB in melanoma.

SERPINB9 is commonly amplified and high expression in cancer cells correlates with poor immune checkpoint blockade response.

Immunotherapies, in particular immune checkpoint blockade (ICB), have improved the clinical outcome of cancer patients, although many fail to mount a durable response. Several resistance mechanisms have been identified, but our understanding of the requirements for a robust ICB response is incomplete. We have engineered an MHC I/antigen: TCR-matched panel of human NSCLC cancer and T cells to identify tumor cell-intrinsic T cell resistance mechanisms. The top differentially expressed gene in resistant tumor cells was SERPINB9. This serine protease inhibitor of the effector T cell-derived molecule granzyme B prevents caspase-mediated tumor apoptosis. Concordantly, we show that genetic ablation of SERPINB9 reverts T cell resistance of NSCLC cell lines, whereas its overexpression reduces T cell sensitivity. SERPINB9 expression in NSCLC strongly correlates with a mesenchymal phenotype. We also find that SERPINB9 is commonly amplified in cancer, particularly melanoma in which it is indicative of poor prognosis. Single-cell RNA sequencing of ICB-treated melanomas revealed that SERPINB9 expression is elevated not only in cells from post- versus pre-treatment cancers, but also in ICB-refractory cancers. In NSCLC we commonly observed rare SERPINB9-positive cancer cells, possibly accounting for reservoirs of ICB-resistant cells. While underscoring SERPINB9 as a potential target to combat immunotherapy resistance, these results suggest its potential to serve as a prognostic and predictive biomarker.

A cellular hierarchy in melanoma uncouples growth and metastasis.

Although melanoma is notorious for its high degree of heterogeneity and plasticity1,2, the origin and magnitude of cell-state diversity remains poorly understood. Equally, it is unclear whether growth and metastatic dissemination are supported by overlapping or distinct melanoma subpopulations. Here, by combining mouse genetics, single-cell and spatial transcriptomics, lineage tracing and quantitative modelling, we provide evidence of a hierarchical model of tumour growth that mirrors the cellular and molecular logic underlying the cell-fate specification and differentiation of the embryonic neural crest. We show that tumorigenic competence is associated with a spatially localized perivascular niche, a phenotype acquired through an intercellular communication pathway established by endothelial cells. Consistent with a model in which only a fraction of cells are fated to fuel growth, temporal single-cell tracing of a population of melanoma cells with a mesenchymal-like state revealed that these cells do not contribute to primary tumour growth but, instead, constitute a pool of metastatic initiating cells that switch cell identity while disseminating to secondary organs. Our data provide a spatially and temporally resolved map of the diversity and trajectories of melanoma cell states and suggest that the ability to support growth and metastasis are limited to distinct pools of cells. The observation that these phenotypic competencies can be dynamically acquired after exposure to specific niche signals warrant the development of therapeutic strategies that interfere with the cancer cell reprogramming activity of such microenvironmental cues.

Plexin-A4 Mediates Cytotoxic T-cell Trafficking and Exclusion in Cancer.

Cytotoxic T cell (CTL) infiltration of the tumor carries the potential to limit cancer progression, but their exclusion by the immunosuppressive tumor microenvironment hampers the efficiency of immunotherapy. Here, we show that expression of the axon guidance molecule Plexin-A4 (Plxna4) in CTLs, especially in effector/memory CD8+ T cells, is induced upon T-cell activation, sustained in the circulation, but reduced when entering the tumor bed. Therefore, we deleted Plxna4 and observed that Plxna4-deficient CTLs acquired improved homing capacity to the lymph nodes and to the tumor, as well as increased proliferation, both achieved through enhanced Rac1 activation. Mice with stromal or hematopoietic Plxna4 deletion exhibited enhanced CTL infiltration and impaired tumor growth. In a melanoma model, adoptive transfer of CTLs lacking Plxna4 prolonged survival and improved therapeutic outcome, which was even stronger when combined with anti-programmed cell death protein 1 (PD-1) treatment. PLXNA4 abundance in circulating CTLs was augmented in melanoma patients versus healthy volunteers but decreased after the first cycle of anti-PD-1, alone or in combination with anti-cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4), in those patients showing complete or partial response to the treatment. Altogether, our data suggest that Plxna4 acts as a "checkpoint," negatively regulating CTL migration and proliferation through cell-autonomous mechanisms independent of the interaction with host-derived Plxna4 ligands, semaphorins. These findings pave the way toward Plxna4-centric immunotherapies and propose Plxna4 detection in circulating CTLs as a potential way to monitor the response to immune checkpoint blockade in patients with metastatic melanoma.

Epithelial-to-mesenchymal-like transition events in melanoma.

Epithelial-to-mesenchymal transition (EMT), a process through which epithelial tumor cells acquire mesenchymal phenotypic properties, contributes to both metastatic dissemination and therapy resistance in cancer. Accumulating evidence indicates that nonepithelial tumors, including melanoma, can also gain mesenchymal-like properties that increase their metastatic propensity and decrease their sensitivity to therapy. In this review, we discuss recent findings, illustrating the striking similarities-but also knowledge gaps-between the biology of mesenchymal-like state(s) in melanoma and mesenchymal state(s) from epithelial cancers. Based on this comparative analysis, we suggest hypothesis-driven experimental approaches to further deepen our understanding of the EMT-like process in melanoma and how such investigations may pave the way towards the identification of clinically relevant biomarkers for prognosis and new therapeutic strategies.